Cross-kingdom mimicry of the receptor signaling and leukocyte recruitment activity of a human cytokine by its plant orthologs

Abstract

Human macrophage migration-inhibitory factor (MIF) is an evolutionarily-conserved protein that has both extracellular immune-modulating and intracellular cell-regulatory functions. MIF plays a role in various diseases, including inflammatory diseases, atherosclerosis, autoimmunity, and cancer. It serves as an inflammatory cytokine and chemokine, but also exhibits enzymatic activity. Secreted MIF binds to cell-surface immune receptors such as CD74 and CXCR4. Plants possess MIF orthologs but lack the associated receptors, suggesting functional diversification across kingdoms. Here, we characterized three MIF orthologs (termed MIF/d-dopachrome tautomerase–like proteins or MDLs) of the model plant Arabidopsis thaliana. Recombinant Arabidopsis MDLs (AtMDLs) share similar secondary structure characteristics with human MIF, yet only have minimal residual tautomerase activity using either p-hydroxyphenylpyruvate or dopachrome methyl ester as substrate. Site-specific mutagenesis suggests that this is due to a distinct amino acid difference at the catalytic cavity-defining residue Asn-98. Surprisingly, AtMDLs bind to the human MIF receptors CD74 and CXCR4. Moreover, they activate CXCR4-dependent signaling in a receptor-specific yeast reporter system and in CXCR4-expressing human HEK293 transfectants. Notably, plant MDLs exert dose-dependent chemotactic activity toward human monocytes and T cells. A small molecule MIF inhibitor and an allosteric CXCR4 inhibitor counteract this function, revealing its specificity. Our results indicate cross-kingdom conservation of the receptor signaling and leukocyte recruitment capacities of human MIF by its plant orthologs. This may point toward a previously unrecognized interplay between plant proteins and the human innate immune system.

Chemokine-like MDL proteins modulate flowering time and innate immunity in plants

“…Surprisingly, the three MDLs can bind to the human MIF receptors CD74 and CXCR4 (which are absent from plants), activate signaling activities downstream of these in human immune cells, and substitute for human MIF in leukocyte recruitment. These findings disclose cross-kingdom mimicry of human MIF by these plant orthologs and reflect their (partial) functional conservation (21,22). Reminiscent of the situation of human immune cells and some parasitic pathogens (19,23), plant-feeding aphids secrete a MIF ortholog into J o u r n a l P r e -p r o o f host cells to suppress plant immune responses, which is necessary for their survival and nourishment (24).…”

“…Similarly, the release of aphid MIFs into a plant host has been demonstrated to modulate plant immunity (24). Apart from the latter report, a comprehensive in silico analysis (20), recombinant protein work (21,50), and phylogenetic studies (18), plant MIF proteins have not been subject to functional investigation, in particular not in their native organismal context. Despite the sequence-relatedness to their human counterparts, MDL proteins thus belong to the still large group of Arabidopsis proteins with yet unknown biochemical and biological function (51).…”

“…In addition, MIF engagement of its chemokine receptors CXCR2 and CXCR4 has been suggested to occur at low nanomolar concentrations at which the monomer would be the favored species (6,10). Although an in silico comparison insinuates also a trimeric structure for the MDLs (20,21), their specific oligomeric species in planta remains unclear.…”

“…For the production of MDL2-specific monoclonal antibodies, highly purified recombinant MDL2 protein was prepared using Rosetta (DE3)-competent E. coli cells, which expressed a pET21-derived gene construct to yield a C-terminally 6xHis-tagged MDL2 (MDL2-6xHis) protein product. Bacterial expression and protein purification were carried out essentially as described previously (21,50). Wistar rats were immunized with 60 µg purified full-length MDL2-6xHis, 5 nmol CpG (TIB MOLBIOL, Berlin, Germany), and an equal volume of Incomplete Freund's adjuvant (IFA; Sigma, St. Louis, USA)).…”

“…MDL3 shows in addition co-expression with a number of prominent genes involved in plant immunity (20). Structure prediction and preliminary experimental data suggest that all three Arabidopsis MDL proteins resemble the secondary and tertiary structure of human MIF (20,21).…”